A One-Step Extraction and Luminescence Assay for Quantifying Glucose and ATP Levels in Cultured HepG2 Cells

International Journal of Molecular Sciences

Viktória Temesfői¹  Tamás Kőszegi¹  Rita Csepregi¹  Nikolett Sali¹  Miklós Poór²  Paul W. Needs³  Paul A. Kroon³ 

[1] Department of Laboratory Medicine, Medical School, University of Pécs, Ifjúság u. 13, H-7624 Pécs, Hungary;János Szentágothai Research Center, Ifjúság u. 20, H-7624 Pécs, Hungary;Quadram Institute Bioscience, Norwich Research Park, Norwich NR4 7UA, UK;

关键词: Intracellular glucose assay;    luminescence;    validation;    ATP;    metabolic inhibitors;    flow cytometry;   

DOI  :  10.3390/ijms19092670

来源: DOAJ

【 摘 要 】

A fluorescence-based enzymatic microplate intracellular glucose assay was designed and fully validated. The method was tested in a hepatocellular cancer cell line (HepG2). Our novel one-step extraction reagent gave stable cell lysates for glucose, adenosine triphosphate (ATP), and total protein determination from the same sample. Limit of detection for glucose was 0.13 µM (26 pmol/well), which is superior to commercially available glucose assays. Both intra- and interday assay imprecision in HepG2 cultures were less than 12% coefficient of variance (CV). In cell lysates spiked with glucose, recovery at two levels varied between 83.70% and 91.81%, and both linearity and stability were acceptable. HepG2 cells treated with agents affecting glucose uptake/metabolism (phloretin, quercetin, quercetin-3′-sulfate, NaF, 3-bromopyruvate, NaN3, oligomycin A, ochratoxin A, cytochalasin B, and anti-GLUT1 antibody) showed dose-dependent changes in glucose and ATP levels without total protein (cell) loss. Finally, we performed flow cytometric glucose uptake measurement in the treated cells using 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose fluorescent glucose analog. Glucose uptake did not always mirror the intracellular glucose levels, which most likely reflects the differences between the two methodologies. However, interpreting data obtained by both methods and taking ATP/protein levels at the same time, one can get information on the mode of action of the compounds.

【 授权许可】

Unknown